HPLC purity vs mass spec identity: what each assay actually proves

If a certificate lists only one of these, it is incomplete in a way that matters. HPLC tells you how clean a sample is. Mass spectrometry tells you what it is. Neither substitutes for the other, and the distinction is not academic — it is the difference between a pure powder and the right pure powder.

HPLC: separation, then counting

Reverse-phase HPLC pushes the sample through a C18 column with a gradient of increasing acetonitrile. Molecules partition between the stationary phase and the mobile phase according to hydrophobicity, so they elute at different times. A detector at ~220 nm records the signal over time, and integrating the peaks gives area percentages — the number sold as purity.

The limitation is structural. HPLC quantifies what the detector sees, and the detector cannot see everything. A compound that co-elutes with the main peak is invisible to it, so a "99.5%" result can still be a mixture. What HPLC does exceptionally well is separating, quantifying and letting you see the trace — a clean single symmetric peak is meaningful evidence even when the exact percentage is method-dependent.

Mass spec: weighing the molecule

Electrospray ionisation transfers the peptide into the gas phase in charged droplets; as they evaporate, the molecules emerge protonated. The instrument then measures mass-to-charge ratio. The result is a fingerprint specific to the compound, and the definitive check is the observed molecular ion against the theoretical mass computed from the sequence.

For large peptides you will often see a doubly charged ion, where the molecule carries two protons and appears at roughly half its mass. That is not an error — it is often the most intense peak, because charge separation improves transmission.

• Adducts. Sodium or potassium replaces a proton, adding ~22 Da or ~38 Da. Expected, and subtractable.
• Deamidation. Glutamine or asparagine loses ammonia, about 17 Da. A real degradation pathway worth watching.
• Oxidation. Methionine gains 16 Da. Common in methionine-containing peptides, including growth-hormone secretagogues such as GHRP-2.

Why both are required

Consider two different failures. A sample of the right peptide contaminated with a peptide sequence error would pass a purity check — one clean peak, 99.8% — while being useless. Conversely, a sample of entirely the wrong compound, synthesised cleanly, would also pass purity at 99.8% while failing identity completely. Only the pair rules out both.

There is a third question neither answers: what the material will do in a system. That is why the third-party panel exists — endotoxin, residual solvents, heavy metals, sterility. Every lot we release is verified against all of them, using the methods in our third-party testing.

To see both numbers side by side, open the COA tab on any catalogue.